US7622552B2 - Ligation method - Google Patents

Ligation method Download PDF

Info

Publication number
US7622552B2
US7622552B2 US10/567,403 US56740304A US7622552B2 US 7622552 B2 US7622552 B2 US 7622552B2 US 56740304 A US56740304 A US 56740304A US 7622552 B2 US7622552 B2 US 7622552B2
Authority
US
United States
Prior art keywords
oligopeptide
moiety
molecule
precursor
providing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US10/567,403
Other languages
English (en)
Other versions
US20060247417A1 (en
Inventor
Graham Cotton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Almac Sciences Scotland Ltd
Original Assignee
Almac Sciences Scotland Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0318276A external-priority patent/GB0318276D0/en
Priority claimed from GB0320122A external-priority patent/GB0320122D0/en
Application filed by Almac Sciences Scotland Ltd filed Critical Almac Sciences Scotland Ltd
Assigned to CSS-ALBACHEM LIMITED reassignment CSS-ALBACHEM LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COTTON, GRAHAM
Assigned to ALMAC SCIENCES (SCOTLAND) LIMITED reassignment ALMAC SCIENCES (SCOTLAND) LIMITED CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: CSS-ALBACHEM LIMITED
Publication of US20060247417A1 publication Critical patent/US20060247417A1/en
Application granted granted Critical
Publication of US7622552B2 publication Critical patent/US7622552B2/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids

Definitions

  • This application relates to a method of ligating two, or more molecules, for example, small organic molecules, labels, peptides etc.
  • a method of ligating a peptide such as ligation of a synthetic peptide to a recombinant peptide.
  • Protein engineering methodologies have proven to be invaluable for generating protein based tools for application in basic research, diagnostics, drug discovery and as protein therapeutics.
  • the ability to manipulate the primary structure of a protein in a controlled manner opens up many new possibilities in the biological and medical sciences. As a consequence, there is a concerted effort on developing methodologies for the site-specific modification of proteins and their subsequent application.
  • proteins having site-specific modifications and/or labels is more readily achievable using chemical synthesis methods.
  • the chemical synthesis of proteins enables multiple modifications to be incorporated into both side-chain and backbone moieties of the protein in a site-specific manner, but, in general, the maximum size of sequence that can be synthesised and isolated is circa 50-100 amino acids.
  • a further approach to the generation of proteins is protein/peptide ligation.
  • mutually reactive chemical functionalities are incorporated at the N- and C-termini of unprotected polypeptide fragments such that when they are mixed, they react in a chemoselective manner to join the two sequences together (Cotton G J and Muir T W. Chem. Biol., 1999, 6, R247-R254).
  • the principle of chemical ligation is shown schematically in FIG. 1 .
  • reaction between a hydrazide and an aldehyde to form a hydrazone (Gaertner H F et al, et al Bioconj. Chem., 1992, 3, 262-268) reaction of an aminoxy group and an aldehyde to form an oxime (Rose K. J. Am. Chem. Soc., 1994, 116, 30-33), reaction of azides and aryl phosphines to form an amide bond (Staudinger ligation) (Nilsson B L, Kiessling L L, and Raines R T. Org. Lett., 2001, 3, 9-12, Kiick et al Proc. Natl.
  • the native chemical ligation method has proved popular, it requires an N-terminal cysteine containing peptide for the reaction and thus, if a cysteine is not present at the appropriate position in the protein, a cysteine needs to be introduced at the ligation site.
  • the introduction of extra thiol groups into a protein sequence may be detrimental to its structure/function, especially since cysteine has a propensity to form disulfide bonds which may disrupt the folding pathway or compromise the function of the folded protein.
  • Protein ligation technologies that enable both synthetic and recombinantly derived protein fragments to be joined together have been described. This enables large proteins to be constructed from combinations of synthetic and recombinant fragments, allowing proteins to be site-specifically modified with both natural and unnatural entities. By utilising such so-called protein semi-synthesis, many different synthetic moieties can be site-specifically incorporated at multiple different sites within a target protein.
  • the recombinant fragments must contain the appropriate reactive functionalities to facilitate ligation.
  • One approach to introduce a unique reactive functionality into a recombinant protein has been through the periodate oxidation of N-terminal serine containing sequences. Such treatment converts the N-terminal serine into a glyoxyl moiety, which contains an N-terminal aldehyde. Synthetic hydrazide containing peptides have then been ligated to the N-terminus of these proteins in a chemoselective manner through hydrazone bond formation with the protein N-terminal glyoxyl group (Gaertner H F et al, et al Bioconj.
  • the first step in protein splicing involves an N ⁇ S (or N ⁇ O) acyl shift in which the N-extein unit is transferred to the sidechain SH or OH group of a conserved Cys/Ser/Thr residue, always located at the immediate N-terminus of the intein. Insights into this mechanism have led to the design of a number of mutant inteins which can only promote the first step of protein splicing (Chong et al Gene. 1997, 192, 271-281, (Noren et al., Angew. Chem. Int. Ed. Engl., 2000, 39, 450-466).
  • Proteins expressed as in frame N-terminal fusions to one of these engineered inteins can be cleaved by thiols via an intermolecular transthioesterification reaction, to generate the recombinant protein C-terminal thioester derivative ( FIG. 3 ) (Chong et al Gene. 1997, 192, 271-281, (Noren et al., Angew. Chem. Int. Ed. Engl., 2000, 39, 450-466) (New England Biolabs Impact System WO 00/18881, WO 0047751).
  • Peptide sequences containing an N-terminal cysteine residue can then be specifically ligated to the C-termini of such recombinant C-terminal thioester proteins (Muir et al Proc. Natl. Acad. Sci. USA., 1998, 95, 6705-6710, Evans Jr et al. Prot. Sci., 1998, 7, 2256-2264), in a procedure termed expressed protein ligation (EPL) or intein-mediated protein ligation (IPL).
  • EPL expressed protein ligation
  • IPL intein-mediated protein ligation
  • the chemoselective ligation of N-terminal cysteine containing peptides to C-terminal thioester containing peptides is performed typically at slightly basic pH and in the presence of a thiol cofactor.
  • the strategy also requires a cysteine to be introduced at the ligation site, if one is not suitably positioned within the primary sequence.
  • the chemokine RANTES is unstable in a buffer of 100 mM NaCl, 100 mM sodium phosphate pH 7.4 containing 100 mM 2-mercaptoethanesulfonic acid (MESNA); a buffer typically used for the ligation of C-terminal thioester molecules to N-terminal cysteine containing molecules (expressed protein ligation and native chemical ligation).
  • MESNA 2-mercaptoethanesulfonic acid
  • RANTES contains two disulphide bonds critical for maintaining the structure and function of the protein.
  • the folded protein was found to be converted within 48 hours to a mixture of the reduced protein and MESNA protein adducts. The majority of the protein mixture subsequently formed a precipitate, presumably reflecting the unfolded nature of these species (Cotton, unpublished).
  • ligation reactions that require thiol containing buffers are, in general, not suitable for maintaining the integrity of disulphide bond containing proteins, such as antibodies, antibody fragments and antibody domains, cytokines, growth factors etc.
  • ligation approaches that are typically performed in the absence of thiols.
  • RANTES was stable in 100 mM NaCl, 100 mM sodium phosphate buffer pH 7.4 and 100 mM sodium acetate buffer pH 4.5 (inventor's unpublished results).
  • Ligation reactions that can be performed under such conditions should therefore be applicable for both disulphide and non-disulphide containing proteins.
  • protein ligation means the joining together of two peptide/protein fragments but this is synonymous with protein labelling whereby the label is a peptide or derivatised peptide.
  • the label is a peptide or derivatised peptide.
  • a small non-peptidic synthetic molecule contains the necessary reactive chemical functionality for protein ligation, then ligation of the synthetic molecule directly to either the N- or C-termini of the protein affords site-specific labelling of the protein.
  • technologies developed for the ligation of protein fragments can also be used for the direct labelling of either the N- or C-termini of peptides or proteins in a site-specific manner irrespective of their sequence.
  • Recombinant proteins containing N-terminal glyoxyl functions have been site-specific N-terminally labelled through reaction with hydrazide or aminoxy derivatives of the label (Geoghegan K F and Stroh J G. Bioconj Chem., 1992, 3, 138-146, Alouni S et al. Eur. J. Biochem., 1995, 227, 328-334).
  • Also recombinant proteins containing N-terminal cysteine residues have been N-terminally labelled through reaction with labels containing thioester functionalities, the label being the acyl substituent of the thioester (Schuler B and Pannell L K. Bioconjug. Chem., 2002, 13, 1039-43) and aldehyde functionalities (Zhao et al. Bioconj. Chem., 1999, 10, 424-430) to form amides and thiazolidines respectively.
  • the present inventors have overcome a number of problems associated with the prior art and have developed a new method for ligating peptide molecules which overcomes a number of the problems of the prior art.
  • a method of producing an oligopeptide product comprising the steps:
  • step (c) where said oligopeptides are linked via a linking moiety having Formula II and where said activated ester moiety of step (b) is not a thioester, said activated ester is a terminal activated ester moiety.
  • linking moieties are linked via a linking moiety having Formula I or Formula III.
  • peptide Unless the context demands otherwise, the terms peptide, oligopeptide, polypeptide and protein are used interchangeably.
  • the activated ester moiety of the first oligopeptide may be any suitable activated ester moiety, such as a thioester moiety, a phenolic ester moiety, an hydroxysuccinimide moiety, or an O-acylisourea moiety.
  • the activated ester moiety is a thioester moiety.
  • Any suitable thioester peptides wherein the peptide is the acyl substituent of the thioester may be used in the present invention ( FIG. 4 ).
  • Such thioester peptides may be synthetically or recombinantly produced.
  • the skilled person is well aware of methods known in the art for generating synthetic peptide thioesters.
  • synthetic peptide thioesters may be produced via synthesis on a resin that generates a C-terminal thioester upon H F cleavage (Hojo et al, Bull. Chem. Soc. Jpn., 1993, 66, 2700-2706).
  • ‘safety catch’ linkers has proved to be popular for generating C-terminal thioesters through thiol induced resin cleavage of the assembled peptide (Shin Y et al, J. Am. Chem. Soc., 1999, 121, 11684-11689).
  • Recombinant C-terminal thioester proteins may be produced by manipulating a naturally occurring biological phenomenon known as protein splicing.
  • protein splicing is a post-translational process in which a precursor protein undergoes a series of intramolecular rearrangements which result in precise removal of an internal region, referred to as an intein, and ligation of the two flanking sequences, termed exteins.
  • the second oligopeptide is generated by thiol reagent induced cleavage of an intein fusion protein.
  • a method of producing an oligopeptide product comprising the steps:
  • the reactive moiety of the first oligopeptide may be any suitable reactive moiety.
  • the reactive moiety is a hydrazine moiety, an amino-oxy moiety or a hydrazide moiety having general formula IV, V or VI respectively.
  • the reactive moiety has Formula IV and, in the oligopeptide product produced by the method of the invention, the first and second oligopeptides are linked via a linking moiety having Formula I.
  • the reactive moiety has Formula V and, in the oligopeptide product produced by the method of the invention, the first and second oligopeptides are linked via a linking moiety having Formula II.
  • the reactive moiety has Formula VI and, in the oligopeptide product produced by the method of the invention, the first and second oligopeptides are linked via a linking moiety having Formula III.
  • the first oligopeptide comprises a reactive moiety, which, in preferred embodiments, may be a hydrazine moiety (e.g. Formula IV), an amino-oxy moiety (e.g. Formula V) or an hydrazide moiety (e.g. Formula VI).
  • a reactive moiety which, in preferred embodiments, may be a hydrazine moiety (e.g. Formula IV), an amino-oxy moiety (e.g. Formula V) or an hydrazide moiety (e.g. Formula VI).
  • a particular advantage of the ligation method of the invention is that it may be performed in the absence of thiols. This enables efficient ligation of proteins/peptides comprising disulphide bonds as well as of proteins without such bonds.
  • At least one of the first and second oligopeptides comprises one or more disulphide bonds.
  • Hydrazine, hydrazide or aminooxy containing derivatives of synthetic oligopeptides may be readily produced using known methods, for example, solid phase synthesis techniques.
  • proteins fused N-terminal to an intein domain can be cleaved from the intein by hydrazine treatment in a selective manner to liberate the desired protein as its corresponding hydrazide derivative (for example, see FIG. 5 ).
  • the first oligopeptide is generated by reaction of hydrazine with an oligopeptide molecule comprising the first oligopeptide fused N-terminal to an intein domain.
  • a third aspect of the invention provides a method of generating a protein hydrazide, said method comprising the steps:
  • the present invention further extends to a “one-step” process for ligating two peptides to generate an oligopeptide product.
  • This may be achieved by reacting a suitable protein linked N-terminal to an intein directly with a polypeptide having a hydrazine, hydrazide or amino-oxy moiety.
  • the invention provides a method of producing an oligopeptide product, the method comprising the steps:
  • the ligation technology of the present invention can thus utilise both synthetic and recombinant proteins and peptides. It thus enables the ligation of two or more synthetic peptides, the ligation of two or more recombinant peptides or the ligation of at least one synthetic peptide with at least one recombinant peptide.
  • the present invention may be used for the labelling of synthetic or recombinant peptides.
  • a method of labelling an oligopeptide comprising the steps:
  • step (c) where said label molecule and the oligopeptide are linked via a linking moiety having Formula II and where said activated ester moiety of step (b) is not a thioester, said activated ester is a terminal activated ester moiety.
  • step (b) the oligopeptide is generated by thiol induced cleavage of an intein fusion protein.
  • a method of labelling an oligopeptide comprising the steps:
  • a label molecule having a terminal activated ester moiety may be used to label an oligopeptide having a reactive moiety.
  • step (c) wherein, in step (c), where said label molecule and the oligopeptide are linked via a linking moiety having Formula II and where said activated ester moiety of step (b) is not a thioester, said activated ester is a terminal activated ester moiety.
  • an oligopeptide present as a precursor molecule linked to an intein molecule may be labelled directly.
  • an eighth aspect of the present invention provides a method of labelling an oligopeptide, the method comprising the steps:
  • label molecules known to the skilled person may be used in methods of the invention.
  • the choice of label will depend on the use to which the labelled peptide is to be put.
  • labels which may be used in the methods of the invention may include fluorophores, crosslinking reagents, spin labels, affinity probes, imaging reagents, for example radioisotopes, chelating agents such as DOTA, polymers such as PEG, lipids, sugars, cytotxic agents, and solid surfaces and beads.
  • At least one of the label and oligopeptides comprises one or more disulphide bonds.
  • the methods of the invention are particularly useful in the ligation of peptides, in particular the ligation of peptides, which, using conventional ligation techniques, would require various protecting groups.
  • the inventors have shown that the methods of the invention may be performed under pH conditions in which only the reactive moieties will react.
  • step (c) of the method is performed at a pH in the range pH 4.0 to pH 8.5, preferably pH 4.0 to 8.0, for example, pH 4.0 to 7.5, more preferably in the range pH 5.0 to pH 8.0, more preferably in the range pH 6.0 to pH 7.5, most preferably in the range pH 6.5 to pH 7.5.
  • the inventors have demonstrated that synthetic peptide C-terminal thioesters specifically react with hydrazine under aqueous conditions at pH 6.0 to form the corresponding peptide hydrazide.
  • This allows ligation methods as described herein to be performed at pH 6.0, without the need for a potentially harmful thiol cofactor (useful if either fragment or final construct is thiol sensitive) and does not lead to the introduction of potentially reactive side-chain groups (such as a thiol) into the protein.
  • synthetic peptide C-terminal thioesters specifically react with hydroxylamine under aqueous conditions at pH 6.0 and pH 6.8 to form the corresponding peptide hydroxamic acid.
  • both synthetic peptide C-terminal thioesters and recombinant protein C-terminal thioesters specifically react with O-methylhydroxylamine under aqueous conditions at pH 7.5, to form the corresponding C-terminal N-methoxy amide derivatives. This allows ligation methods as described herein to be performed at pH 7.5, without the need for a potentially harmful thiol cofactor.
  • Peptides and proteins that contain thioester groups can be reacted with hydrazine, hydrazide or aminooxy derivatives of a label or a peptide to afford site-specific labelling and chemoselective ligation respectively (see, for example, FIGS. 4 and 5 ).
  • peptides that contain hydrazine, hydrazide or aminooxy groups can be reacted with thioester derivatives of a label or a peptide to afford site-specific labelling and chemoselective ligation respectively (see, for example, FIGS. 4 and 5 ).
  • protein hydrazides can be generated by cleavage of protein-intein fusions with hydrazine
  • the inventors have shown that such protein hydrazides may be ligated by reaction of the hydrazide moiety with reactive groups other than activated ester moieties, for example an aldehyde functionality or a ketone functionality.
  • a pyruvoyl derivative of a synthetic peptide can be chemoselectively ligated to the C-terminus of recombinant protein hydrazides using the described approach, and in an analogous fashion, a pyruvoyl derivative of fluorescein was used to site-specifically label the C-terminus of recombinant protein hydrazides using the described approach.
  • This aspect of the invention provides a further novel method of ligating a recombinant peptide to a second peptide or indeed a label.
  • a ninth aspect of the invention provides a method of producing an oligopeptide product, the method comprising the steps:
  • FIG. 6 An example of this aspect is shown in FIG. 6 .
  • a tenth aspect of the invention provides a method of labelling an oligopeptide, the method comprising the steps:
  • the hydrazone moiety has Formula VII:
  • R is H or any substituted or unsubstituted, preferably unsubstituted, alkyl group.
  • the method is performed at a pH in the range pH 1.0 to pH 7.0, preferably pH 1.0 to pH 6.0, more preferably in the range pH 2.0 to pH 5.5, most preferably in the range pH 2.0 to pH 4.5.
  • the aldehyde or ketone containing moiety of the oligopeptide or of the label is an ⁇ -diketone group or an ⁇ -keto aldehyde group.
  • an oligopeptide product produced using a method of the invention.
  • a labelled oligopeptide comprising an oligopeptide labelled according to a method of the invention.
  • FIG. 1 illustrates schematically the general principle of chemical ligation.
  • FIG. 2 illustrates schematically the mechanism of protein splicing.
  • FIG. 3 illustrates the generation of recombinant C-terminal thioester proteins.
  • FIG. 4 illustrates ligation of protein and peptide thioesters with hydrazine and aminooxy containing entities, such as labels, peptides and proteins.
  • FIG. 5 illustrates the generation of synthetic and recombinant peptide hydrazides for ligation with thioester containing molecules. Note the peptide or label is is the acyl substituent of the thioester.
  • FIG. 6 illustrates the generation of recombinant peptide hydrazides for ligation with aldehyde and ketone containing molecules.
  • FIG. 7 illustrates SDS-PAGE analysis of Grb2-SH2-GyrA-CBD (immobilised on chitin beads) treated with DTT and MESNA.
  • Molecular weight markers (lane 1); purified Grb2-SH2-GyrA-CBD immobilised on chitin beads (lane 4).
  • Grb2-SH2-GyrA-CBD treated with 100 mM DTT (lanes 5 and 7) or 120 mM MESNA (lanes 8 and 10). Both the whole reaction slurries (lanes 5 and 8) and the reaction supernatants (lanes 7 and 10) were analysed.
  • FIG. 8 illustrates SDS-PAGE analysis of Grb2-SH2-GyrA-CBD (immobilised on chitin beads) treated with hydrazine.
  • Molecular weight markers (lane 1);
  • Grb2-SH2-GyrA-CBD immobilised on chitin beads after 20 h treatment with phosphate buffer only (lane 2).
  • Grb2-SH2-GyrA-CBD treated with 200 mM hydrazine in phosphate buffer for 20 h. The whole reaction slurries were analysed.
  • FIG. 9 illustrates an ESMS spectrum of the C-terminal hydrazide derivative of Grb2-SH2.
  • FIG. 10 shows SDS-PAGE analysis of the reaction between synthetic ketone containing peptide CH 3 COCO-myc with Grb2-SH2-C-terminal hydrazide and Cytochrome C.
  • Molecular weight markers (lane 1); Grb2-SH2-C-terminal DTT thioester (lane 2).
  • FIG. 11 shows the structure of CH 3 COCO-Lys(F1). The 5-carboxy fluorescein positional isomer is shown.
  • FIG. 12 illustrates SDS-PAGE analysis of the reaction between CH 3 COCO-Lys(F1) with Grb2-SH2 C-terminal hydrazide in 50 mM sodium acetate buffer pH 4.5.
  • FIG. 13 illustrates SDS-PAGE analysis of the reaction between CH 3 COCO-Lys(F1) with Cytochrome C in 100 mM sodium acetate buffer pH 4.5.
  • Molecular weight markers (lane 1); Cytochrome C (lane 2).
  • FIG. 14 illustrates SDS-PAGE analysis of the reaction of CH 3 COCO-Lys(F1) with Grb2-SH2 C-terminal hydrazide and with Cytochrome C in 50 mM sodium acetate buffer pH 4.5.
  • A total protein stain of gel. Prior to this coomassie staining (A), the gel was imaged for green fluorescence (B).
  • B green fluorescence
  • FIG. 15 shows SDS-PAGE analysis of the reaction between CH 3 COCO-Lys(F1) and Grb2 SH2 C-terminal hydrazide in 40% aqueous acetonitrile containing 0.1% TFA; reaction after 4 h (lane 1), 24 h (lane 2), 48 h (lane 3), Grb2 SH2 C-terminal hydrazide (lane 4).
  • AS626p1A 200 mM sodium phosphate buffer pH 6.0 containing 100 mM hydrazine monohydrate (200 ⁇ L) was added to a model synthetic C-terminal thioester peptide termed AS626p1A (200 ⁇ g) to yield a final peptide concentration of 317 ⁇ M.
  • AS626p1A has sequence ARTKQ TARK(Me) 3 STGGKAPRKQ LATKAARK-COS-(CH 2 ) 2 —COOC 2 H 5 (SEQ ID NO: 1) wherein a single Alanine residue (which may be any one of the Alanine residues of SEQ ID NO: 1) is substituted by an Arginine residue.
  • the reaction was incubated at room temperature and monitored with time by analytical reversed phase HPLC.
  • Vydac C18 column (5 ⁇ M, 0.46 ⁇ 25 cm). Linear gradients of acetonitrile water/0.1% TFA were used to elute the peptides at a flow rate of 1 mL min ⁇ 1 . Individual peptides eluting from the column were characterised by electrospray mass spectrometry.
  • the C-terminal mercaptoethanesulfonic acid thioester derivative of recombinant Grb2-SH2 was generated through cleavage of the fusion protein Grb2-SH2-GyrA intein-CBD as described in Example 2 below.
  • This recombinant C-terminal thioester protein (100 ⁇ g) was reacted with 100 mM O-methylhydroxylamine in 200 mM sodium phosphate buffer pH 7.5 (200 ⁇ L). The reaction was incubated at room temperature and monitored with time by analytical reversed phase HPLC. Vydac C5 column (5 ⁇ M, 0.46 ⁇ 25 cm).
  • Linear gradients of acetonitrile water/0.1% TFA were used to elute the peptides at a flow rate of 1 mL min ⁇ 1 .
  • Individual peptides eluting from the column were characterised by electrospray mass-spectrometry.
  • the peptide C-terminal thioester was converted to the corresponding peptide hydroxamic acid in a clean fashion with no side-product formation.
  • Increasing the pH of the reaction buffer accelerated the rate of reaction. For instance, with a concentration of 100 mM NH 2 OH, on moving from pH 6.0 to pH 6.8 the percentage product formation after 1 h increased from 25% to 91%.
  • the rate of reaction with 100 mM NH 2 OH atpH 6.0 was comparable with 10 mM NH 2 OH at pH 6.8.
  • the rate of reaction of the peptide C-terminal thioester with hydroxalymine, to form the corresponding hydroxamic acid increases with increasing pH and decreases with decreasing NH 2 OH concentrations.
  • the labelling was performed under increasing pH and decreasing NH 2 OH concentrations.
  • the purified synthetic 27 amino acid C-terminal thioester peptide (ethyl 3-mercaptopropionate thioester, observed mass 3155 Da) was incubated at room temperature with 100 mM D-methylhydroxylamine in 200 mM sodium phosphate buffer pH 7.5.
  • the peptide C-terminal thioester reacted to form a single product that eluted earlier than the starting thioester peptide as analysed by reverse phase HPLC.
  • This material corresponded to the expected N-methoxy peptide amide as determined by ESMS: observed mass 3070 Da, expected mass 3068 Da.
  • the kinetics of the reaction were monitored using reverse phase HPLC (Table II).
  • the peptide C-terminal thioester was converted to the corresponding N-methoxy peptide amide derivative in a clean fashion with no side-product formation, with the reaction 75% complete after 24 h. Under these conditions no thioester hydrolysis was observed.
  • lysis buffer 0.1 mM EDTA, 250 mM NaCl, 5% glycerol, 1 mM PMSF, 25 mM HEPES, pH 7.4
  • the soluble fraction was loaded onto a chitin column pre-equilibrated in lysis buffer.
  • the column was then washed with wash buffer (1 mM EDTA, 250 mM NaCl, 0.1% Triton-X 100, 25 mM HEPES, pH 7.0) to yield purified Grb2-SH2-GyrA-CBD immobilised on chitin beads ( FIG. 7 ).
  • Chitin beads containing immobilised Grb2-SH2-GyrA-CBD were therefore equilibrated into 200 mM NaCl, 200 mM phosphate buffer pH 7.4 and hydrazine monohydrate added in the same buffer to give a 50% slurry with a final hydrazine concentration of 200 mM.
  • the mixture was then rocked at room temperature and analysed by SDS-PAGE ( FIG. 8 ). After 20 hours the supernatant was removed and analysed by HPLC and ESMS.
  • Grb2-SH2 C-terminal hydrazide was isolated from the supernatant by either (i) using RPHPLC followed by lyophilisation or (ii) by gel filtration.
  • the Grb2-SH2 C-terminal hydrazide reaction solution was loaded onto a superdex peptide column (Amersham Biosciences) and eluted with a running buffer of 50 mM sodium acetate pH 4.5. This yielded a solution of purified Grb2-SH2 C-terminal hydrazide in 50 mM sodium acetate pH 4.5. This solution was concentrated using a centricon filter (3000 MWCO), then snap frozen and stored at ⁇ 20° C. until use.
  • the expression vector pMYB5 (New England Biolabs) encodes for a fusion protein comprising maltose binding protein (sequence above) fused N-terminal to the Sce VMA intein, which is in turn fused to the N-terminus of a chitin binding domain (CBD) to facilitate purification.
  • CBD chitin binding domain
  • E. coli cells were transformed with this plasmid and grown in LB medium to mid log phase and protein expression induced for 4 h at 37° C. with 0.5 mM IPTG. After centrifugation the cells were re-suspended in lysis buffer (0.1 mM EDTA, 250 mM NaCl, 5% glycerol, 1 mM PMSF, 25 mM HEPES, pH 7.4) and lysed by sonication. The soluble fraction was loaded onto a chitin column pre-equilibrated in lysis buffer.
  • lysis buffer 0.1 mM EDTA, 250 mM NaCl, 5% glycerol, 1 mM PMSF, 25 mM HEPES, pH 7.4
  • the column was then washed with wash buffer (1 mM EDTA, 250 mM NaCl, 0.1% Triton-X 100, 25 mM HEPES, pH 7.0) to yield the purified fusion protein (MBP-VMA-CBD) immobilised on chitin beads.
  • wash buffer (1 mM EDTA, 250 mM NaCl, 0.1% Triton-X 100, 25 mM HEPES, pH 7.0
  • MESNA 2-mercaptoethanesulfonic acid
  • MBP-VMA-CBD fusion with MESNA results in cleavage of the fusion protein into two protein species.
  • the molecular size of the two fragments corresponds to that of the MBP and the VMA-CBD portion, indicative that cleavage has taken place at the MBP-VMA intein junction.
  • Cleavage of the precursor fusion protein liberates MBP into the supernatant while the VMA-CBD portion remains immobilized on the chitin beads. This was confirmed by ESMS analysis of the cleavage supernatant, which contained one protein species.
  • Chitin beads containing immobilised MBP-VMA-CBD were equilibrated into 200 mM NaCl, 200 mM phosphate buffer pH 7.4 and hydrazine monohydrate added in the same buffer to give a 50% slurry with a final hydrazine concentration of 200 mM. The mixture was then rocked at room temperature and analysed by SDS-PAGE and by HPLC and ESMS.
  • MBP C-terminal hydrazide was isolated from the supernatant by either (i) using RPHPLC followed by lyophilisation or (ii) by gel filtration.
  • MBP C-terminal hydrazide reaction solution was loaded onto a superdex peptide column (Amersham Biosciences) and eluted with a running buffer of 50 mM sodium acetate buffer pH 4.5. This yielded a solution of purified MBP C-terminal hydrazide in 50 mM sodium acetate buffer pH 4.5.
  • This protein solution was concentrated using a centricon filter (3000 MWCO), then snap frozen and stored at ⁇ 20° C. until use.
  • MBP-VMA-CBD fusion Treatment of MBP-VMA-CBD fusion with hydrazine results in cleavage of the fusion protein into two species.
  • the molecular size of the two fragments as analysed by SDS-PAGE corresponds to MBP and the VMA-CBD portion, indicative that cleavage has taken place at the unique thioester linkage between the MBP-VMA intein domain.
  • Cleavage of the precursor fusion protein liberates MBP into the supernatant, while the VMA-CBD portion remains immobilized on the chitin beads.
  • HPLC and ESMS analysis of the cleavage supernatant confirms that a single protein species is generated with an observed mass of 42988 Da.
  • the expected mass difference between the C-terminal MESNA thioester derivative of a protein and its corresponding C-terminal hydrazide is 111 Da.
  • the observed mass of the C-terminal MESNA thioester of MBP was found to be 43098 Da.
  • the product from the hydrazine cleavage of MBP-VMA-CBD is 110 Da lower, indicating that the desired C-terminal hydrazide derivative of MBP had been formed.
  • the fluorophore needs to contain the appropriate reactive group for ligation, namely an aldehyde or ketone functionality.
  • a derivative of fluorescein was synthesized containing a pyruvoyl moiety. Initially, Fmoc-Lys(Mtt)-OH was coupled to a rink amide resin, and the Mtt group removed using standard procedures (1% TFA, 4% triisopropylsilane in dichloromethane). 5(6)-carboxyfluorescein was then couple to the lysine ⁇ -amino group.
  • the Fmoc group was then removed and pyruvic acid coupled to the free ⁇ -amino group of the lysine.
  • the desired fluorescein derivative [designated CH 3 COCO-Lys(F1), see FIG. 11 ] was purified to >95% purity by RPHPLC and lyophilised (ESMS, expected monoisotopic mass 576.2 Da; observed monoisotopic mass 576.0 Da).
  • Grb2 SH2 C-terminal hydrazide was converted into a single protein species with an apparent increased molecular weight expected for that of the desired product, and this newly formed protein was green fluorescent when visualised under a UV lamp.
  • ESMS of the reaction product confirmed that one fluoresein molecule had been added to the protein. The reaction is virtually complete after 4 h, with prolonged incubation appearing to be detrimental to the formation of the ligation product.
  • the described approach was used for the site-specific C-terminal labeling of MBP with fluorescein.
  • a sample (250 ⁇ g) of lyophilised recombinant MBP C-terminal hydrazide (generated through hydrazine cleavage of MBP-VMA-CBD precursor fusion protein) was dissolved in 40% aqueous acetonitrile containing 0.1% TFA (200 ⁇ L). The solution was then added to a sample of CH 3 COCO-Lys (F1) to give a final fluorophore concentration of circa 0.3 mM. The reaction was then incubated at room temperature and periodically analyzed by SDS-PAGE.
  • the present invention provides novel methods of protein ligation and protein labelling. These enable both synthetic and recombinantly derived protein fragments to be efficiently joined together in a regioselective manner. This thus enables large proteins to be constructed from combinations of synthetic and recombinant fragments and allows proteins of any size to be site-specifically modified in an unprecedented manner. This is of major importance for biological and biomedical science and drug discovery when one considers that the ⁇ 30,000 human genes yield hundreds of thousands of different protein species through post-translational modification. Such post-translationally modified proteins cannot be accessed through current recombinant technologies.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Surgical Instruments (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Iron Core Of Rotating Electric Machines (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
US10/567,403 2003-08-05 2004-08-05 Ligation method Expired - Lifetime US7622552B2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB0318276.3 2003-08-05
GB0318276A GB0318276D0 (en) 2003-08-05 2003-08-05 Ligation method
GB0320122.5 2003-08-28
GB0320122A GB0320122D0 (en) 2003-08-28 2003-08-28 Ligation method
PCT/GB2004/003391 WO2005014620A1 (fr) 2003-08-05 2004-08-05 Procede de ligature

Publications (2)

Publication Number Publication Date
US20060247417A1 US20060247417A1 (en) 2006-11-02
US7622552B2 true US7622552B2 (en) 2009-11-24

Family

ID=34137749

Family Applications (1)

Application Number Title Priority Date Filing Date
US10/567,403 Expired - Lifetime US7622552B2 (en) 2003-08-05 2004-08-05 Ligation method

Country Status (12)

Country Link
US (1) US7622552B2 (fr)
EP (1) EP1651664B9 (fr)
JP (1) JP4637838B2 (fr)
AT (1) ATE429439T1 (fr)
AU (1) AU2004263366A1 (fr)
CA (1) CA2534520C (fr)
DE (1) DE602004020776D1 (fr)
DK (1) DK1651664T3 (fr)
ES (1) ES2323706T3 (fr)
PL (1) PL1651664T3 (fr)
PT (1) PT1651664E (fr)
WO (1) WO2005014620A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039715A1 (fr) * 2012-09-07 2014-03-13 University Of Rochester Procédés et compositions pour le marquage spécifique d'un site de peptides et de protéines
US10053491B2 (en) * 2013-11-05 2018-08-21 Ajinomoto Co., Inc. Method for producing peptide hydrazide, peptide amide, and peptide thioester

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009508470A (ja) * 2005-07-21 2009-03-05 アボット・ラボラトリーズ Ｓｏｒｆ構築物並びにポリタンパク質、プロタンパク質及びタンパク質分解による方法を含む複数の遺伝子発現
EP1770099A1 (fr) * 2005-09-28 2007-04-04 University of Geneva Procédé pour la production des polypeptides modifiées
EP2350112A4 (fr) * 2008-10-03 2013-01-16 Advanced Proteome Therapeutics Inc Modifications de n-terminaux spécifiques de site de protéines et formation de conjugué
GB0908393D0 (en) * 2009-05-15 2009-06-24 Almac Sciences Scotland Ltd Labelling method
TW201124535A (en) * 2009-10-30 2011-07-16 Abbott Lab SORF constructs and multiple gene expression
US9593142B2 (en) 2013-12-26 2017-03-14 New York University Aldehyde capture ligation technology for synthesis of amide bonds
GB201721802D0 (en) 2017-12-22 2018-02-07 Almac Discovery Ltd Ror1-specific antigen binding molecules
GB202020154D0 (en) 2020-12-18 2021-02-03 Almac Discovery Ltd ROR1-specific variant antigen binding molecules
CN115043899A (zh) * 2022-05-24 2022-09-13 南开大学 双胺或胺与硫醇偶联的化合物及其制备方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3985617A (en) * 1974-10-29 1976-10-12 Ajinomoto Co., Inc. Immobilization of biologically active proteins with a polypeptide azide
US5965714A (en) * 1997-10-02 1999-10-12 Connaught Laboratories, Inc. Method for the covalent attachment of polysaccharides to protein molecules
WO2000018881A2 (fr) 1998-09-30 2000-04-06 New England Biolabs, Inc. Ligation de peptide induite par une inteine
US6194451B1 (en) * 1997-01-31 2001-02-27 Pharmacia & Upjohn S.P.A. Matrix metalloproteinase inhibitors
US20040058390A1 (en) * 2000-06-19 2004-03-25 Zyomyx, Inc. Methods for immobilizing polypeptides
US20040067537A1 (en) * 2000-07-13 2004-04-08 Hahn Klaus M. Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6586398B1 (en) * 2000-04-07 2003-07-01 Amgen, Inc. Chemically modified novel erythropoietin stimulating protein compositions and methods

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3985617A (en) * 1974-10-29 1976-10-12 Ajinomoto Co., Inc. Immobilization of biologically active proteins with a polypeptide azide
US6194451B1 (en) * 1997-01-31 2001-02-27 Pharmacia & Upjohn S.P.A. Matrix metalloproteinase inhibitors
US5965714A (en) * 1997-10-02 1999-10-12 Connaught Laboratories, Inc. Method for the covalent attachment of polysaccharides to protein molecules
WO2000018881A2 (fr) 1998-09-30 2000-04-06 New England Biolabs, Inc. Ligation de peptide induite par une inteine
US20040058390A1 (en) * 2000-06-19 2004-03-25 Zyomyx, Inc. Methods for immobilizing polypeptides
US20040067537A1 (en) * 2000-07-13 2004-04-08 Hahn Klaus M. Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Bonnet et al. Chemoselective acylation of hydrazinopeptides . . . Tetrahedron Letters. 2000, vol. 41, pp. 45-48. *
Chong et al., "Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element", Gene: An International Journal on Genes and Genomes 192(2):271-281 (1997).
Cotton et al., "Peptide ligation and its application to protein engineering", Chemistry and Biology 6(2):R247-R256 (1999).
Geoghegan, K F, "Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine", Bioconjugate Chemistry, American Chemical Society 3(2):138-146 (1992).
Perler et al., "The mechanism of protein splicing: variations on a theme", Peptides 2002, pp. 254-255 (2002).
Salvadori et al. Aminoxy Analogs of Leu-Enkephalin. International Journal of Peptide and Protein Research. 1981, vol. 18, pp. 393-401. *
Vanderesse et al. Alpha-Aminoxy Acids as Building Blocks for the Oxime . . . Journal of Peptide Science. May 2003, vol. 9, Issue 5, pp. 282-299. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039715A1 (fr) * 2012-09-07 2014-03-13 University Of Rochester Procédés et compositions pour le marquage spécifique d'un site de peptides et de protéines
US9557336B2 (en) 2012-09-07 2017-01-31 University Of Rochester Methods and compositions for site-specific labeling of peptides and proteins
US10053491B2 (en) * 2013-11-05 2018-08-21 Ajinomoto Co., Inc. Method for producing peptide hydrazide, peptide amide, and peptide thioester

Also Published As

Publication number Publication date
EP1651664B1 (fr) 2009-04-22
PL1651664T3 (pl) 2009-09-30
AU2004263366A1 (en) 2005-02-17
US20060247417A1 (en) 2006-11-02
PT1651664E (pt) 2009-09-02
ATE429439T1 (de) 2009-05-15
DK1651664T3 (da) 2009-08-24
WO2005014620A1 (fr) 2005-02-17
CA2534520C (fr) 2014-04-08
CA2534520A1 (fr) 2005-02-17
ES2323706T3 (es) 2009-07-23
EP1651664B9 (fr) 2012-03-21
DE602004020776D1 (de) 2009-06-04
JP4637838B2 (ja) 2011-02-23
JP2007534626A (ja) 2007-11-29
EP1651664A1 (fr) 2006-05-03

Similar Documents

Publication Publication Date Title
Tan et al. Chemical protein synthesis: advances, challenges, and outlooks
Spears et al. Cysteine protecting groups: applications in peptide and protein science
McGrath et al. Chemoselectivity in chemical biology: acyl transfer reactions with sulfur and selenium
Hackenberger et al. Chemoselective ligation and modification strategies for peptides and proteins
Malins et al. Peptide ligation chemistry at selenol amino acids
von Eggelkraut-Gottanka et al. Peptide αthioester formation using standard Fmoc-chemistry
US20150152134A1 (en) Protein retrosplicing enabled by a double ligation reaction
Englebretsen et al. A novel thioether linker: chemical synthesis of a HIV-1 protease analogue by thioether ligation
US7622552B2 (en) Ligation method
Hou et al. Progress in chemical synthesis of peptides and proteins
Harmand et al. New chemistries for chemoselective peptide ligations and the total synthesis of proteins
US8859730B2 (en) Native chemical ligation at serine and threonine sites
Laps et al. Harnessing the power of transition metals in solid-phase peptide synthesis and key steps in the (semi) synthesis of proteins
JP4694085B2 (ja) アミド結合を形成するためのライゲーション方法及び試薬
Malins et al. Modern extensions of native chemical ligation for chemical protein synthesis
Li et al. Preparation of peptide selenoesters from their corresponding acyl hydrazides
EP1117693B1 (fr) Ligation de peptide induite par une inteine
Guan et al. Synthetic thiol and selenol derived amino acids for expanding the scope of chemical protein synthesis
US7001745B1 (en) Intein mediated peptide ligation
JP6493216B2 (ja) ペプチドヒドラジド、ペプチドアミド及びペプチドチオエステルの製造方法
Bastings et al. One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester
KR101676515B1 (ko) 펩티드 티오에스테르체의 제조방법
Otaka Development of naturally inspired peptide and protein chemistry
CN114480534B (zh) 基于转肽酶的化学酶法的蛋白质半合成
AU4862801A (en) Purification of polypeptides

Legal Events

Date Code Title Description
AS Assignment

Owner name: CSS-ALBACHEM LIMITED, UNITED KINGDOM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:COTTON, GRAHAM;REEL/FRAME:017412/0466

Effective date: 20060227

AS Assignment

Owner name: ALMAC SCIENCES (SCOTLAND) LIMITED, UNITED KINGDOM

Free format text: CHANGE OF NAME;ASSIGNOR:CSS-ALBACHEM LIMITED;REEL/FRAME:018403/0336

Effective date: 20060613

STCF Information on status: patent grant

Free format text: PATENTED CASE

CC Certificate of correction
FPAY Fee payment

Year of fee payment: 4

FPAY Fee payment

Year of fee payment: 8

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1553); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 12